GSM7089065: mouse IgG rep1; Homo sapiens; RIP-Seq
To separate cells into cytoplasmic fraction and nuclear pellet, 80~90% confluent cells in 150 mm dishes were lysed with HLB/NP40 buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.5% NP40, and 2.5 mM MgCl2) and incubated for 5 min on ice. After the incubation, HLB/NP40 buffer containing 10% sucrose was added under the lysate layer, followed isolation of nuclear pellet by centrifugation. Nuclear fraction was treated with MNase digestion to separate proteins associated with chromatins and treated with NaCl to remove nucleoplasmic proteins.  The final pellet was subjected to sonication and centrifugation, and the supernatant was collected as a nuclear speckle (NS)-enriched fraction. We then isolated hTERT complex by immunoprecipitation with an anti-hTERT mAb from the NS-enriched fraction and conducted RIP-seq analysis for RNAs associated with hTERT in nuclear speckles. Sequencing cDNA libraries (RIP-seq libraries) were prepared using SMARTer stranded Total RNA Sample Prep Kit-HI Mammalian (Takara Bio) according to the manufacturer's instruction.